This invention pertains generally to the field of biology and particularly to apparatus for use in the analysis and sequencing of DNA and related polymers.
The sequencing of deoxyribonucleic acid (DNA) is a fundamental tool of modern biology and is conventionally carried out in various ways, commonly by processes which separate DNA segments by electrophoresis. See, e.g., Current Protocols In Molecular Biology, Vol. 1, Chapter 7, xe2x80x9cDNA Sequencing,xe2x80x9d 1995. The sequencing of several important genomes has already been completed (e.g., yeast, E. coli), and work is proceeding on the sequencing of other genomes of medical and agricultural importance (e.g., human, C. elegans, Arabidopsis). In the medical context, it will be necessary to xe2x80x9cre-sequencexe2x80x9d the genome of large numbers of human individuals to determine which genotypes are associated with which diseases. Such sequencing techniques can be used to determine which genes are active and which inactive either in specific tissues, such as cancers, or more generally in individuals exhibiting genetically influenced diseases. The results of such investigations can allow identification of the proteins that are good targets for new drugs or identification of appropriate genetic alterations that may be effective in genetic therapy. Other applications lie in fields such as soil ecology or pathology where it would be desirable to be able to isolate DNA from any soil or tissue sample and use probes from ribosomal DNA sequences from all known microbes to identify the microbes present in the sample.
The conventional sequencing of DNA using electrophoresis is typically laborious and time consuming. Various alternatives to conventional DNA sequencing have been proposed. One such alternative approach, utilizing an array of oligonucleotide probes synthesized by photolithographic techniques is described in Pease, et al., xe2x80x9cLight-Generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis,xe2x80x9d Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5022-5026, May 1994. In this approach, the surface of a solid support modified with photolabile protecting groups is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions. A 3xe2x80x2 activated deoxynucleoside, protected at the 5xe2x80x2 hydroxyl with a photolabile group, is then provided to the surface such that coupling occurs at sites that had been exposed to light. Following capping, and oxidation, the substrate is rinsed and the surface is illuminated through a second mask to expose additional hydroxyl groups for coupling. A second 5xe2x80x2 protected activated deoxynucleoside base is presented to the surface. The selective photodeprotection and coupling cycles are repeated to build up levels of bases until the desired set of probes is obtained. It may be possible to generate high density miniaturized arrays of oligonucleotide probes using such photolithographic techniques wherein the sequence of the oligonucleotide probe at each site in the array is known. These probes can then be used to search for complementary sequences on a target strand of DNA, with detection of the target that has hybridized to particular probes accomplished by the use of fluorescent markers coupled to the targets and inspection by an appropriate fluorescence scanning microscope. A variation of this process using polymeric semiconductor photoresists, which are selectively patterned by photolithographic techniques, rather than using photolabile 5xe2x80x2 protecting groups, is described in McGall, et al., xe2x80x9cLight-Directed Synthesis of High-Density Oligonucleotide Arrays Using Semiconductor Photoresists,xe2x80x9d Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 13555-13560, November 1996, and G. H. McGall, et al., xe2x80x9cThe Efficiency of Light-Directed Synthesis of DNA Arrays on Glass Substrates,xe2x80x9d Journal of the American Chemical Society 119, No. 22, 1997, pp. 5081-5090.
A disadvantage of both of these approaches is that four different lithographic masks are needed for each monomeric base, and the total number of different masks required are thus four times the length of the DNA probe sequences to be synthesized. The high cost of producing the many precision photolithographic masks that are required, and the multiple processing steps required for repositioning of the masks for every exposure, contribute to relatively high costs and lengthy processing times.
An improved process for synthesizing arrays of DNA probe sequences, polypeptides, and the like, rapidly and efficiently by a patterning process utilizing a computer controlled image former, is described in published PCT application International Publication No. WO 99/42813, published Aug. 26, 1999, entitled Method and Apparatus for Synthesis of Arrays of DNA Probes. This process eliminates the need for a lithographic mask, significantly reducing the costs and time delays that have been associated with processes requiring such masks. In the patterning process described in the foregoing published PCT application, a substrate with an active surface to which, e.g., DNA synthesis linkers have been applied, is used to support probes to be activated. To activate the surface a high precision two-dimensional light image is projected onto the substrate by an image former, illuminating those pixels on the active surface which are to be activated to bind a first base. The light incident on the pixels in the array to which the light is applied deprotects OH groups and makes them available for binding the bases. After this development step, a fluid containing the appropriate base is provided to the active surface of the substrate and the selected base binds to the exposed sites. The process is repeated until all of the elements of the two-dimensional array on the substrate surface have an appropriate base bound thereto. The process is repeated for other pixel locations and desired levels of bases until the entire selected two-dimensional array of probe sequences has been completed. To provide the various chemicals in an appropriate sequence to the substrate, the substrate may be mounted within a flow cell having an enclosure which seals off the active surface of the substrate, allowing the appropriate reagents to flow through the flow cell and over the active surface.
The present invention is directed to an improved flow cell of the type that may be utilized in the synthesis of arrays of DNA probe sequences, polypeptides and the like, and is particularly adapted to be used with image formers for projecting an array of patterned light onto a substrate held by the flow cell. The flow cell of the invention is formed to precisely align a substrate with respect to an image former while distributing the fluid containing the appropriate chemicals through the active volume and over the active exposed surface of the flow cell, while minimizing the total volume of fluid contained within the flow cell to conserve the reagents being utilized. The flow cell allows fast and simple removal and replacement of substrates while insuring a tight seal around the substrate to minimize the leakage of reagents in the flow cell, and it locates the active surface of the substrate at the focal plane of the image former with a high degree of accuracy and repeatability.
A flow cell of a preferred construction in accordance with the invention includes a base having a central window opening and a registration surface against which a substrate may be mounted with its active surface opposite to that which is engaged against the registration surface. A gasket having a central opening defining an active area surrounded by the material of the gasket is mounted on the active surface of the substrate. The gasket has inlet and outlet extension openings which optionally and preferably extend away from the central opening in the gasket. A press block has an engagement surface which is adapted to the engaged against the gasket to fully enclose an active volume between the press block, the peripheral walls of the central opening in the gasket, and the active surface of the substrate. A press mounted to the base is formed to selectively press the press block against the gasket and hold it in position. The press block preferably includes a channel therein which extends from an exterior surface of the press block to a position in communication with the inlet opening extension in the gasket and another channel extending from the exterior surface of the press block to communication with the outlet extension opening in the gasket, thereby allowing reagents to flow into the active volume between the inlet opening extension and the outlet opening extension across the active surface area defined by the central opening in the gasket. The registration surface of the base is preferably raised above adjacent areas of the base and surrounds the central window opening to define a flat plane. The plane of the registration surface is utilized to precisely locate the active surface of the substrate with respect to an optical image former which projects an image through the window opening of the base and through a transparent substrate to a focal plane at the active surface of the substrate. The gasket is preferably formed of a thin non-reactive material having parallel flat surfaces. The thin gasket allows the active volume within the flow cell through which reagents flow to be minimized, with the extension openings in the gasket allowing inlet and outlet of the reagent into and out of the active volume in a manner which allows substantially the full central opening area of the gasket to be utilized as the active volume, with uniform flow of reagent across the active volume.
A press structure is preferably utilized to selectively press the press block against the gasket and the substrate. The press structure includes a standing frame secured to the base and having an upright section and an arm section which extends therefrom over the central opening in the base. A press screw is threadingly engaged with the arm and has a drive end positioned to engage an external surface of the press block as the press screw is turned to thread it toward the press block. The drive end is preferably rounded and fits into a rounded concave depression in the press block to provide a ball-and-socket engagement between the press screw and the press block that allows the press block to seat against the gasket and provide even pressure by the press block over the entire surface area of the gasket. When a substrate is to be changed, the press screw can be easily unscrewed by the operator until the press block is free of the press screw, allowing the press block and substrate to be removed, a new substrate to be inserted into position and the gasket and press block repositioned onto the active substrate, after which the press screw can be threaded down into contact with the press block to drive it into engagement with the gasket to seal the active volume in the flow cell.
Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.